B cell receptor associated proteins

ABSTRACT

The present invention provides B cell receptor associated proteins (BCRP) and polynucleotides which identify and encode BCRP. The invention also provides expression vectors, host cells, agonists, antibodies, and antagonists. The invention also provides methods for treating disorders associated with expression of BCRP.

[0001] This application is a divisional of copending U.S. applicationSer. No. 08/851,971, filed on May 7, 1997, entitled B CELL RECEPTORASSOCIATED PROTEINS.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof B cell receptor associated proteins and to the use of these sequencesin the diagnosis, prevention, and treatment of immunological disordersand cancer.

BACKGROUND OF THE INVENTION

[0003] The B-cell response to antigens, which is modulated throughreceptors, is an essential component of the normal immune system.Immature B cells undergo a selection process based on antigen bindingprior to leaving the bone marrow. Mature B cells recognize foreignantigens through B cell receptors (BCR) and produce specific antibodieswhich bind the foreign antigens. To generate an efficient response tocomplex antigens, the BCR, BCR associated proteins, and T cellassistance are required. The antigen/receptor complex is internalized,and the antigen is proteolytically processed. A small part of theantigen remains complexed with major histocompatability complex-II(MHCII) molecules on the surface of the B cells where the complex can berecognized by T cells. T cells activated by antigen presentation secretea variety of lymphokines that induce B cell maturation.

[0004] Signaling through the BCR plays an important role in both thegeneration of antibody and in the establishment of immunologicaltolerance. Immature B cells that bind self-antigens while still in thebone marrow are eliminated by apoptosis. In contrast, antigen binding onmature B cells results in activation, proliferation, anergy, orapoptosis. The particular functional response depends on whether the Bcell receives co-stimulatory signals through other surface receptors andwhich signal transduction pathways are activated. A mature B cellco-expresses two classes of membrane-bound immunoglobin, IgM and IgD,which have identical cytoplasmic domains and identical antigen bindingspecificity. In order to be expressed on the B cell surface, the BCR,whether it is of the IgM or IgD class, must be associated with two otherpolypeptides, Ig-α and Ig-β. The cytoplasmic portions of theIgM/Ig-α/Ig-β and the IgD/Ig-α/Ig-β complex should therefore beidentical to each other.

[0005] Both Ig-α and Ig-β contain a sequence, ITAM, that can causeactivation of protein tyrosine kinases. In addition, the cytoplasmictails of Ig-α and Ig-β are sufficient for the internalization of thesurface proteins and their targeting to endosomal compartments forprocessing. Early biochemical events in signal transduction, such asprotein kinase activation and release of calcium ions, are similar forthe two receptors (IgM and IgD); their subsequent biological effects,however, are different. Antigen binding or cross-linking of the IgMreceptor leads to apoptosis, while binding of IgM and IgD, or IgD alone,does not. Binding to IgD alone induces cell proliferation. Analysis ofIgD-deficient mice shows that the absence of IgD reduces the efficacy ofB cell participation in immune responses. Further in vitro differencesin antibody responses, immunological memory, and tolerance have alsobeen described (Carsetti, R. et al. (1993) Eur. J. Immunol. 23: 168-178;Roes, J. et al. (1993) J. Expt. Med. 177: 45-55; and Kim, K. M. et al.(1992) J. Immunol. 148: 29-34).

[0006] Additional analysis of the receptor complex shows that at leastfive more proteins are associated with the mouse BCR. B cell associatedproteins (BAPs) with a mass of 31, 37, and 41 kDa are specificallyassociated with IgM, while 29 and 31 kDa BAPs are preferentiallyassociated with IgD. BAP 41 has not been characterized, but BAP 32 andBAP 37 are related to prohibitin, a protein which has been implicated inthe control of cell proliferation and may have tumor suppressoractivity. Both BAP 32 and 37 interact with IgM via its transmembranedomain and contain a C-terminal NPXY motif associated withinternalization of proteins. The IgD-associated BAP 29 and BAP 31 showstructural features suggesting that they co-localize with the membraneIgM BCR in the plasma membrane. Northern analysis shows that BAP 29, BAP31, BAP 32, and BAP 37 are expressed in all tissue types examined, andthat BAP 32 and BAP 37 are expressed more strongly in transformed celllines than in normal tissues (Kim, K. -M., et al. (1994) EMBO J.13:3793-3800; and Terashima, M., et al. (1994) EMBO J.13: 3782-3792).

[0007] The discovery of new B-cell receptor associated proteins and thepolynucleotides encoding them satisfies a need in the art by providingnew compositions which are useful in the diagnosis, prevention andtreatment of cancer and immunological disorders.

SUMMARY OF THE INVENTION

[0008] The present invention features a B-cell receptor-associatedprotein hereinafter designated BCRP1 and characterized as havingsimilarity to mouse BAP 29.

[0009] Accordingly, the invention features a substantially purifiedBCRP1 having the amino acid sequence shown in SEQ ID NO:1.

[0010] One aspect of the invention features isolated and substantiallypurified polynucleotides that encode BCRP1. In a particular aspect, thepolynucleotide is the nucleotide sequence of SEQ ID NO:2.

[0011] The invention also relates to a polynucleotide sequencecomprising the complement of SEQ ID NO:2 or variants thereof. Inaddition, the invention features polynucleotide sequences whichhybridize under stringent conditions to SEQ ID NO:2.

[0012] The present invention additionally features a B-cellreceptor-associated protein hereinafter designated BCRP2 andcharacterized as having similarity to mouse BAP 37.

[0013] Accordingly, the invention features a substantially purifiedBCRP2 having the amino acid sequence shown in SEQ ID NO:3. The inventionalso features a polypeptide variant of BCRP2, SEQ ID NO:5, where F₉₁ andF₉₆ are replaced by S, and V₁₂₆ is replaced by L.

[0014] One aspect of the invention features isolated and substantiallypurified polynucleotides that encode BCRP2 and the BCRP2 variant (SEQ IDNO:4 and SEQ ID NO:6).

[0015] The invention also features polynucleotide sequences comprisingthe complement of SEQ ID NO:4, SEQ ID NO:6, or variants thereof. Inaddition, the invention features polynucleotide sequences whichhybridize under stringent conditions to SEQ ID NO:4 and SEQ ID NO:6.

[0016] The invention additionally features fragments or portions of thenucleic acid sequences encoding the claimed polypeptides, and expressionvectors and host cells comprising polynucleotides that encode BCRP. Thepresent invention also features antibodies which bind specifically toBCRP, and pharmaceutical compositions comprising substantially purifiedBCRP. The invention also features the use of agonists and antagonists ofBCRP. The invention also features a method for treating cancer usingantagonists of BCRP1, BCRP2, and agonists of BCRP2; for treatingimmunological disorders using antagonists of BCRP1; and for treatingdisorders associated with cell growth and differentiation using BCRP1.

BRIEF DESCRIPTION OF THE FIGURES

[0017]FIGS. 1A, 1B, and 1C show the amino acid sequence (SEQ ID NO:1)and nucleic acid sequence (SEQ ID NO:2) of BCRP1. The alignment wasproduced using MACDNASIS PRO software (Hitachi Software Engineering Co.,Ltd., San Bruno, Calif.).

[0018]FIGS. 2A, 2B, 2C and 2D show the amino acid sequence (SEQ ID NO:3)and nucleic acid sequence (SEQ ID NO:4) of BCRP2. The alignment wasproduced using MACDNASIS PRO software (Hitachi Software Engineering Co.,Ltd., San Bruno, Calif.).

[0019]FIG. 3 shows the amino acid sequence alignments between BCRP1 (SEQID NO:1) and mouse BAP 29 (GI 541730, SEQ ID NO:7). The alignment wasproduced using the multisequence alignment program of DNASTAR software(DNASTAR Inc, Madison Wis.).

[0020]FIGS. 4A and 4B show the amino acid sequence alignments betweenBCRP2 (SEQ ID NO:3), BCRP2 variant (SEQ ID NO:5) and mouse BAP 37 (GI541734, SEQ ID NO:8). The alignment was produced using the multisequencealignment program of DNASTAR software (DNASTAR Inc, Madison Wis.).

DESCRIPTION OF THE INVENTION

[0021] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0022] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

[0023] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

[0024] Definitions

[0025] “Nucleic acid sequence”, as used herein refers to anoligonucleotide, nucleotide, or polynucleotide, and fragments orportions thereof, and to DNA or RNA of genomic or synthetic origin whichmay be single- or double-stranded, and represent the sense or complementor antisense strand. Similarly, “amino acid sequence” as used hereinrefers to an oligopeptide, peptide, polypeptide, or protein sequence,and fragments or portions thereof, and to naturally occurring orsynthetic molecules.

[0026] Where “amino acid sequence” is recited herein to refer to anamino acid sequence of a naturally occurring protein molecule, “aminoacid sequence” and like terms, such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

[0027] “Peptide nucleic acid”, as used herein, refers to a moleculewhich comprises an oligomer to which an amino acid residue, such aslysine, and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen, P. E. et al. (1993)Anticancer Drug Des. 8:53-63).

[0028] BCRP, as used herein, refers to the amino acid sequences ofsubstantially purified BCRP obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

[0029] “Consensus”, as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, or which has beenextended using the XL-PCR kit (Perkin Elmer, Norwalk, Conn.) in the 5′and/or the 3′ direction and resequenced, or which has been assembledfrom the overlapping sequences of more than one Incyte clone using theGELVIEW fragment assembly system (GCG, Madison, Wis.), or which has beenboth extended and assembled.

[0030] A “variant” of BCRP, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

[0031] A “deletion”, as used herein, refers to a change in either aminoacid or nucleotide sequence in which one or more amino acid ornucleotide residues, respectively, are absent.

[0032] An “insertion” or “addition”, as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid or nucleotide residues, respectively, as compared tothe naturally occurring molecule.

[0033] A “substitution”, as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0034] The term “biologically active”, as used herein, refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic BCRP, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0035] The term “agonist”, as used herein, refers to a molecule which,when bound to BCRP, causes a change in BCRP which modulates the activityof BCRP. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to BCRP.

[0036] The terms “antagonist” or “inhibitor”, as used herein, refer to amolecule which, when bound to BCRP, blocks or modulates the biologicalor immunological activity of BCRP. Antagonists and inhibitors mayinclude proteins, nucleic acids, carbohydrates, or any other moleculeswhich bind to BCRP.

[0037] The term “modulate”, as used herein, refers to a change or analteration in the biological activity of BCRP. Modulation may be anincrease or a decrease in protein activity, a change in bindingcharacteristics, or any other change in the biological, functional orimmunological properties of BCRP.

[0038] The term “mimetic”, as used herein, refers to a molecule, thestructure of which is developed from knowledge of the structure of BCRPor portions thereof and, as such, is able to effect some or all of theactions of BCRP-like molecules.

[0039] The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding BCRP or the encoded BCRP.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

[0040] The term “substantially purified”, as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

[0041] “Amplification”, as used herein refers to the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0042] The term “hybridization”, as used herein, refers to any processby which a strand of nucleic acid binds with a complementary strandthrough base pairing.

[0043] The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

[0044] The terms “complementary” or “complementarity”, as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands.

[0045] The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term“substantially homologous.”The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

[0046] As known in the art, numerous equivalent conditions may beemployed to comprise either low or high stringency conditions. Factorssuch as the length and nature (DNA, RNA, base composition) of thesequence, nature of the target (DNA, RNA, base composition, presence insolution or immobilization, etc.), and the concentration of the saltsand other components (e.g., the presence or absence of formamide,dextran sulfate and/or polyethylene glycol) are considered and thehybridization solution may be varied to generate conditions of eitherlow or high stringency different from, but equivalent to, the abovelisted conditions.

[0047] The term “stringent conditions”, as used herein, is the“stringency” which occurs within a range from about Tm−5° C. (5° C.below the melting temperature (Tm) of the probe) to about 20° C. to 25°C. below Tm. As will be understood by those of skill in the art, thestringency of hybridization may be altered in order to identify ordetect identical or related polynucleotide sequences.

[0048] The term “antisense”, as used herein, refers to nucleotidesequences which are complementary to a specific DNA or RNA sequence. Theterm “antisense strand” is used in reference to a nucleic acid strandthat is complementary to the “sense” strand. Antisense or complementarymolecules may be produced by any method, including synthesis by ligatingthe gene(s) of interest in a reverse orientation to a viral promoterwhich permits the synthesis of a complementary strand. Once introducedinto a cell, this transcribed strand combines with natural sequencesproduced by the cell to form duplexes. These duplexes then block eitherthe further transcription or translation. In this manner, mutantphenotypes may be generated. The designation “negative” is sometimesused in reference to the antisense or complement strand, and “positive”is sometimes used in reference to the sense strand.

[0049] The term “portion”, as used herein, with regard to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from four amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ IDNO:1” encompasses the full-length human BCRP and fragments thereof.

[0050] “Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

[0051] The term “antigenic determinant”, as used herein, refers to thatportion of a molecule that makes contact with a particular antibody(i.e., an epitope). When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0052] The terms “specific binding” or “specifically binding”, as usedherein, in reference to the interaction of an antibody and a protein orpeptide, mean that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words, the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A”, the presence of aprotein containing epitope A (or free, unlabeled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

[0053] The term “sample”, as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acid encoding BCRPor fragments thereof may comprise a cell, chromosomes isolated from acell (e.g., a spread of metaphase chromosomes), genomic DNA (in solutionor bound to a solid support such as for Southern analysis), RNA (insolution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

[0054] The term “correlates with expression of a polynucleotide”, asused herein, indicates that the detection of the presence of ribonucleicacid that is similar to SEQ ID NO:2 by northern analysis is indicativeof the presence of mRNA encoding BCRP in a sample and thereby correlateswith expression of the transcript from the polynucleotide encoding theprotein.

[0055] “Alterations” in the polynucleotide of SEQ ID NO:2, as usedherein, comprise any alteration in the sequence of polynucleotidesencoding BCRP including deletions, insertions, and point mutations thatmay be detected using hybridization assays. Included within thisdefinition is the detection of alterations to the genomic DNA sequencewhich encodes BCRP (e.g., by alterations in the pattern of restrictionfragment length polymorphisms capable of hybridizing to SEQ ID NO:2),the inability of a selected fragment of SEQ ID NO:2 to hybridize to asample of genomic DNA (e.g., using allele-specific oligonucleotideprobes), and improper or unexpected hybridization, such as hybridizationto a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding BCRP (e.g., using fluorescent in situhybridization [FISH] to metaphase chromosomes spreads).

[0056] As used herein, the term “antibody” refers to intact molecules aswell as fragments thereof, such as Fab, F(ab′)₂, and Fv, which arecapable of binding the epitopic determinant. Antibodies that bind BCRPpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or peptide used to immunize an animal can be derived fromthe transition of RNA or synthesized chemically, and can be conjugatedto a carrier protein, if desired. Commonly used carriers that arechemically coupled to peptides include bovine serum albumin andthyroglobulin. The coupled peptide is then used to immunize the animal(e.g., a mouse, a rat, or a rabbit).

[0057] The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

[0058] The Invention

[0059] The invention is based on the discovery of two B-cell receptorassociated proteins, (BCRP1 and BCRP2) and a variant of BCRP2,collectively referred to as BCRP, the polynucleotides encoding BCRP, andthe use of these compositions for the diagnosis, prevention, ortreatment of disorders associated with the expression of BCRP.

[0060] Nucleic acids encoding the human BCRP1 of the present inventionwere first identified in Incyte Clone 1383303 from the brain tumor cDNAlibrary (BRAITUT08) through a computer search for amino acid sequencealignments. A consensus sequence, SEQ ID NO:2, was derived from thefollowing overlapping and/or extended nucleic acid sequences: IncyteClones; 1383303, 1385136 (BRAITUT08), 1002194 (BRSTNOT03), 1392554(THYRNOT03), 1480614 (CORPNOT02), 1631701 (COLNNOT19), 462896(LATRNOT01), 531583 (BRAINOT03), and 967961 (BRSTNOT05).

[0061] Nucleic acids encoding the human BCRP2 of the present inventionwere first identified in Incyte Clone 484722 from the teratocarcinomacDNA library (HNT2RAT01) through a computer search for amino acidsequence alignments. A consensus sequence, SEQ ID NO:4, was derived fromthe following overlapping and/or extended nucleic acid sequences: IncyteClones; 484722 (HNT2RAT01), 1354868 (LUNGNOT09), 161583 (ADENINB01),272498 (LIVRNOT02), 344519 (THYMNOT02), 041573 (TBLYNOT01), 458853(KERANOT01), 687606 (UTRSNOT02), 786831 (PROSNOT05), 871306 (LUNGAST01),932480 (CERVNOT01), and 980758 (TONGTUT01).

[0062] Nucleic acids encoding the human BCRP2 variant of the presentinvention were first identified in Incyte Clone 454790 from thekeratinocyte cell cDNA library (KERANOT01) through a computer-generatedsearch for amino acid sequence alignments. A consensus sequence, SEQ IDNO:6, was derived from extension and assembly of nucleic acid sequencesof Incyte Clone 454790 (KERANOT01), 1354868 (LUNGNOT09), 161583(ADENINB01), 272498 (LIVRNOT02), 344519 (THYMNOT02), 041573 (TBLYNOT01),458853 (KERANOT01), 687606 (UTRSNOT02), 786831 (PROSNOT05), 871306(LUNGAST01), 932480 (CERVNOT01), and 980758 (TONGTUT01).

[0063] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A,1B and IC. BCRP1 is 241 amino acids in length and has two potentialcAMP/cGMP-dependent protein kinase sites; R₇₁-S₇₄ and R₁₉₂-S₁₉₅. BCRP1contains a leucine zipper-like motif comprising L₁₇₃, L₁₈₀, L₁₈₇, L₁₉₁,L₁₉₈, M₂₀₈, L₂₁₅, L₂₂₂, and L₂₂₉ and a C-terminal NPXY motif associatedwith internalization of proteins. BCRP1 has chemical and structuralhomology with mouse BAP29 (GI 541730; SEQ ID NO:7). In particular, BCRP1and mouse BAP29 share 78% identity. Northern analysis shows theexpression of this sequence in various libraries, a majority of whichare associated with cancers and proliferating cells and tissues.

[0064] In another embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:3, as shown in FIGS. 2A,2B, 2C, and 2D. BCRP2 is 299 amino acids in length and contains apotential N glycosylation site at N₁₄₉ and two potential protein kinaseC phosphorylation sites at T₁₅₅ and T₁₆₉. BCRP2 has chemical andstructural homology with mouse BAP37 (SEQ ID NO:8), in particular, BCRP2shares 97% identity with mouse BAP37.

[0065] The invention also encompasses BCRP variants, including thevariant shown in SEQ ID NO:5. A preferred BCRP variant is one having atleast 80%, and more preferably 90%, amino acid sequence identity to theBCRP1 amino acid sequence (SEQ ID NO:1) or the BCRP2 amino acid sequence(SEQ ID NO:3). A most preferred BCRP variant is one having at least 95%amino acid sequence identity to SEQ ID NO:1 or SEQ ID NO:3.

[0066] The invention also encompasses polynucleotides which encode BCRP.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of BCRP can be used to generate recombinant molecules whichexpress BCRP. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 asshown in FIGS. 1A, 1B, and 1C, and SEQ ID NO:4 as shown in FIGS. 2A, 2B,2C, and 2D.

[0067] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding BCRP, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring BCRP, and all such variations are to beconsidered as being specifically disclosed.

[0068] Although nucleotide sequences which encode BCRP and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring BCRP under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding BCRP or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding BCRP and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0069] The invention also encompasses production of DNA sequences, orportions thereof, which encode BCRP and its derivatives, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art at the time of thefiling of this application. Moreover, synthetic chemistry may be used tointroduce mutations into a sequence encoding BCRP or any portionthereof.

[0070] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed nucleotide sequences, andin particular, those shown in SEQ ID NO:2, under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Wahl, G. M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) andKimmel, A. R. (1987; Methods Enzymol. 152:507-511), and may be used at adefined stringency.

[0071] Altered nucleic acid sequences encoding BCRP which areencompassed by the invention include deletions, insertions, orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent BCRP. The encodedprotein may also contain deletions, insertions, or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent BCRP. Deliberate amino acid substitutions may bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the biological activity of BCRP is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline; glycine and alanine; asparagine and glutamine; serine andthreonine; phenylalanine and tyrosine.

[0072] Also included within the scope of the present invention arealleles of the genes encoding BCRP. As used herein, an “allele” or“allelic sequence” is an alternative form of the gene which may resultfrom at least one mutation in the nucleic acid sequence. Alleles mayresult in altered mRNAs or polypeptides whose structure or function mayor may not be altered. Any given gene may have none, one, or manyallelic forms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0073] Methods for DNA sequencing which are well known and generallyavailable in the art may be used to practice any embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE (US Biochemical Corp, Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of recombinant polymerases andproofreading exonucleases such as the ELONGASE amplification systemmarketed by Gibco BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the MICROLAB 2200 liquid transfer system(Hamilton, Reno, Nev.), PTC200 thermal cycler (MJ Research, Watertown,Mass.) and the ABI 377 DNA sequencers (Perkin Elmer).

[0074] The nucleic acid sequences encoding BCRP may be extendedutilizing a partial nucleotide sequence and employing various methodsknown in the art to detect upstream sequences such as promoters andregulatory elements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to linker sequence and a primer specific to the knownregion. The amplified sequences are then subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

[0075] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO4.06 primer analysis software (National Biosciences Inc., Plymouth,Minn.), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

[0076] Another method which may be used is capture PCR which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR.

[0077] Another method which may be used to retrieve unknown sequences isthat of Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries (Clontech, Palo Alto, Calif.) to walk in genomic DNA. Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions. When screening for full-length cDNAs, it ispreferable to use libraries that have been size-selected to includelarger cDNAs. Also, random-primed libraries are preferable, in that theywill contain more sequences which contain the 5′ regions of genes. Useof a randomly primed library may be especially preferable for situationsin which an oligo d(T) library does not yield a full-length cDNA.Genomic libraries may be useful for extension of sequence into the 5′and 3′ non-transcribed regulatory regions.

[0078] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled device camera. Output/light intensity may be converted toelectrical signal using appropriate software (e.g. GENOTYPER andSEQUENCE NAVIGATOR, Perkin Elmer) and the entire process from loading ofsamples to computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable for thesequencing of small pieces of DNA which might be present in limitedamounts in a particular sample.

[0079] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode BCRP, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of BCRP in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and expressBCRP.

[0080] As will be understood by those of skill in the art, it may beadvantageous to produce BCRP-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

[0081] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterBCRP encoding sequences for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, or introduce mutations, and so forth.

[0082] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding BCRP may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of BCRP activity, it may be useful toencode a chimeric BCRP protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the BCRP encoding sequence and theheterologous protein sequence, so that BCRP may be cleaved and purifiedaway from the heterologous moiety.

[0083] In another embodiment, sequences encoding BCRP may besynthesized, in whole or in part, using chemical methods well known inthe art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of BCRP, or a portion thereof. Forexample, peptide synthesis can be performed using various solid-phasetechniques (Roberge, J. Y. et al. (1995) Science 269:202-204) andautomated synthesis may be achieved, for example, using the ABI 431Apeptide synthesizer (Perkin Elmer).

[0084] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of BCRP, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0085] In order to express a biologically active BCRP, the nucleotidesequences encoding BCRP or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0086] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding BCRPand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

[0087] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding BCRP. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0088] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding BCRP,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

[0089] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for BCRP. For example, whenlarge quantities of BCRP are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding BCRP may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0090] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0091] In cases where plant expression vectors are used, the expressionof sequences encoding BCRP may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

[0092] An insect system may also be used to express BCRP. For example,in one such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding BCRPmay be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of BCRP will render the polyhedrin gene inactiveand produce recombinant virus lacking coat protein. The recombinantviruses may then be used to infect, for example, S. frugiperda cells orTrichoplusia larvae in which BCRP may be expressed (Engelhard, E. K. etal. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

[0093] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding BCRP may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing BCRP in infected host cells (Logan, J.and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0094] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding BCRP. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding BCRP, its initiation codon, and upstream sequencesare inserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a portion thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

[0095] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

[0096] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress BCRP may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

[0097] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980)Cell 22:817-23) genes which can be employed in tk⁻ or aprt⁻ cells,respectively. Also, antimetabolite, antibiotic or herbicide resistancecan be used as the basis for selection; for example, dhfr, which confersresistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad.Sci. 77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14); and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0098] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding BCRP isinserted within a marker gene sequence, recombinant cells containingsequences encoding BCRP can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding BCRP under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

[0099] Alternatively, host cells which contain the nucleic acid sequenceencoding BCRP and express BCRP may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

[0100] The presence of polynucleotide sequences encoding BCRP can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or portions or fragments of polynucleotides encoding BCRP.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding BCRP todetect transformants containing DNA or RNA encoding BCRP. As used herein“oligonucleotides” or “oligomers” refer to a nucleic acid sequence of atleast about 10 nucleotides and as many as about 60 nucleotides,preferably about 15 to 30 nucleotides, and more preferably about 20-25nucleotides, which can be used as a probe or amplimer.

[0101] A variety of protocols for detecting and measuring the expressionof BCRP, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson BCRP is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

[0102] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding BCRPinclude oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding BCRP, or any portions thereof may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo,Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland,Ohio)). Suitable reporter molecules or labels which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

[0103] Host cells transformed with nucleotide sequences encoding BCRPmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by arecombinant cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode BCRP may be designed to contain signal sequences which directsecretion of BCRP through a prokaryotic or eukaryotic cell membrane.Other recombinant constructions may be used to join sequences encodingBCRP to nucleotide sequence encoding a polypeptide domain which willfacilitate purification of soluble proteins. Such purificationfacilitating domains include, but are not limited to, metal chelatingpeptides such as histidine-tryptophan modules that allow purification onimmobilized metals, protein A domains that allow purification onimmobilized immunoglobulin, and the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp., Seattle, Wash.).The inclusion of cleavable linker sequences such as those specific forFactor XA or enterokinase (Invitrogen, San Diego, Calif.) between thepurification domain and BCRP may be used to facilitate purification. Onesuch expression vector provides for expression of a fusion proteincontaining BCRP and a nucleic acid encoding 6 histidine residuespreceding a thioredoxin or an enterokinase cleavage site. The histidineresidues facilitate purification on IMIAC (immobilized metal ionaffinity chromatography) as described in Porath, J. et al. (1992; Prot.Exp. Purif. 3: 263-281) while the enterokinase cleavage site provides ameans for purifying BCRP from the fusion protein. A discussion ofvectors which contain fusion proteins is provided in Kroll, D. J. et al.(1993; DNA Cell Biol. 12:441-453).

[0104] In addition to recombinant production, fragments of BCRP may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using an ABI 431A peptidesynthesizer (Perkin Elmer). Various fragments of BCRP may be chemicallysynthesized separately and combined using chemical methods to producethe full length molecule.

[0105] Therapeutics

[0106] Based on the chemical and structural homology between BCRP1 (SEQID NO:1) and mouse BAP 29 (SEQ ID NO:7) and between BCRP2 (SEQ ID NO:3)and mouse BAP 37 (SEQ ID NO:8), the BCRPs are B cell receptor associatedproteins which have a role in signal transduction. BCR-associatedproteins control B cell responses by regulating cell differentiation,proliferation, signal transduction, and the internalization andprocessing of the B cell receptor-antigen complex. Proteins with similarsignal transduction functions may be used to modulate cell growth,differentiation, maturation, and cellular responses to lymphokines andother extracellular signals. In Northern analysis, BCRP1 sequences areassociated with cancerous and proliferating cells and tissues, and assuch, may play a role in modulating cell growth and differentiation.

[0107] Therefore, in one embodiment, antagonists or inhibitors of BCRP1may be administered to a subject to treat or prevent cancer. Thesecancers include, but are not limited to, adenocarcinoma, leukemia,lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma andparticularly cancers of the adrenal gland, bladder, bone, brain, breast,cervix, gastrointestinal tract, heart, kidney, liver, lung, ovaries,pancreas, parathyroid, pituitary gland, prostate, salivary gland,spleen, stomach, thymus, thyroid, testes, and uterus.

[0108] In another embodiment, a vector expressing the complement orantisense of the polynucleotide encoding BCRP1 may be administered to asubject treat or prevent the cancers listed above. In one aspect,antibodies which are specific for BCRP may be used directly as anantagonist, or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express BCRP.

[0109] In another embodiment, antagonists or inhibitors of BCRP1 may beadministered to a subject to treat or prevent an immunological disorder.These disorders include, but are not limited to, adult respiratorydistress syndrome, allergies, asthma, atherosclerosis, arteriosclerosis,bronchitis, dermatomyositis, neurofibromatosis, prostate hyperplasia,polymyositis, and rheumatoid arthritis.

[0110] In another embodiment, a vector expressing the complement orantisense of the polynucleotide encoding BCRP1 may be administered to asubject to treat or prevent the disorders of cell proliferation listedin the preceding paragraph.

[0111] In another embodiment, BCRP1 or a fragment or derivative thereofmay be administered to a subject to treat or prevent disordersassociated with cell growth and differentiation. These include, but arenot limited to, Alzheimer's disease, heart attacks, osteoarthritis,osteoporosis, Parkinson's disease, rheumatoid arthritis, stroke, woundhealing, and damage to heart and nerve cells caused by ischemia, freeradicals, and toxins.

[0112] In another embodiment, a vector capable of expressing BCRP1, or afragment or a derivative thereof, may also be administered to a subjectto treat or prevent the disorders associated with cell growth anddifferentiation listed in the preceding paragraph.

[0113] In another embodiment, agonists of BCRP1 may be administered to asubject to treat or prevent the disorders associated with cell growthand differentiation listed above.

[0114] BCRP2 expression may induce apoptosis and may be used toselectively eliminate cells and tissues. Therefore, in one embodiment,BCRP2 or a fragment or derivative thereof may be administered to asubject to treat a disorder of cell growth, including cancer. Thesecancers include, but are not limited to, adenocarcinoma, leukemia,lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma andparticularly cancers of the adrenal gland, bladder, bone, brain, breast,cervix, gastrointestinal tract, heart, kidney, liver, lung, ovaries,pancreas, parathyroid, pituitary gland, prostate, salivary gland,spleen, stomach, thymus, thyroid, testes, and uterus.

[0115] In another embodiment, a vector capable of expressing BCRP2, or afragment or a derivative thereof, may also be administered to a subjectto treat a disorder of cell growth and particularly those listed in thepreceding paragraph.

[0116] In other embodiments, any of the therapeutic proteins,antagonists, antibodies, agonists, antisense or complement sequences orvectors described above may be administered in combination with otherappropriate therapeutic agents. Selection of the appropriate agents foruse in combination therapy may be made by one of ordinary skill in theart, according to conventional pharmaceutical principles. Thecombination of therapeutic agents may act synergistically to effect thetreatment or prevention of the various disorders described above. Usingthis approach, one may be able to achieve therapeutic efficacy withlower dosages of each agent, thus reducing the potential for adverseside effects.

[0117] Antagonists or inhibitors of BCRP may be produced using methodswhich are generally known in the art. In particular, purified BCRP maybe used to produce antibodies or to screen libraries of pharmaceuticalagents to identify those which specifically bind BCRP.

[0118] Antibodies to BCRP may be generated using methods that are wellknown in the art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0119] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith BCRP or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

[0120] It is preferred that the peptides, fragments, or oligopeptidesused to induce antibodies to BCRP have an amino acid sequence consistingof at least five amino acids, and more preferably at least 10 aminoacids. It is also preferable that they are identical to a portion of theamino acid sequence of the natural protein, and they may contain theentire amino acid sequence of a small, naturally occurring molecule.Short stretches of BCRP amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

[0121] Monoclonal antibodies to BCRP may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120).

[0122] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceBCRP-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

[0123] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

[0124] Antibody fragments which contain specific binding sites for BCRPmay also be generated. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

[0125] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between BCRP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering BCRP epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

[0126] In another embodiment of the invention, the polynucleotidesencoding BCRP, or any fragment thereof, or antisense or complementmolecules, may be used for therapeutic purposes. In one aspect,antisense or complement to the polynucleotide encoding BCRP may be usedin situations in which it would be desirable to block the transcriptionof the mRNA. In particular, cells may be transformed with sequencescomplementary to polynucleotides encoding BCRP. Thus, antisense orcomplement molecules may be used to modulate BCRP activity, or toachieve regulation of gene function. Such technology is now well knownin the art, and sense or antisense or complement oligomers or largerfragments, can be designed from various locations along the coding orcontrol regions of sequences encoding BCRP.

[0127] Expression vectors derived from retro viruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensemolecules complementary to the polynucleotides of the gene encodingBCRP. These techniques are described both in Sambrook et al. (supra) andin Ausubel et al. (supra).

[0128] Genes encoding BCRP can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide or fragment thereof which encodes BCRP. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the DNA, suchvectors may continue to transcribe RNA molecules until they are disabledby endogenous nucleases. Transient expression may last for a month ormore with a non-replicating vector and even longer if appropriatereplication elements are part of the vector system.

[0129] As mentioned above, modifications of gene expression can beobtained by designing antisense molecules, DNA, RNA, or PNA, to thecontrol regions of the gene encoding BCRP, i.e., the promoters,enhancers, and introns. Oligonucleotides derived from the transcriptioninitiation site, e.g., between positions −10 and +10 from the startsite, are preferred. Similarly, inhibition can be achieved using “triplehelix” base-pairing methodology. Triple helix pairing is useful becauseit causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (Gee, J. E. et al. (1994) In: Huber, B.E. and B. I. Carr, Molecular and Immunologic Approaches, FuturaPublishing Co., Mt. Kisco, N.Y.). The antisense molecules may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

[0130] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding BCRP.

[0131] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0132] Antisense molecules and ribozymes of the invention may beprepared by any method known in the art for the synthesis of nucleicacid molecules. These include techniques for chemically synthesizingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences encoding BCRP. Such DNA sequences may beincorporated into a wide variety of vectors with suitable RNA polymerasepromoters such as T7 or SP6. Alternatively, these cDNA constructs thatsynthesize antisense RNA constitutively or inducibly can be introducedinto cell lines, cells, or tissues.

[0133] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0134] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection and by liposomeinjections may be achieved using methods which are well known in theart.

[0135] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0136] An additional embodiment of the invention relates to theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of BCRP,antibodies to BCRP, mimetics, agonists, antagonists, or inhibitors ofBCRP. The compositions may be administered alone or in combination withat least one other agent, such as stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

[0137] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0138] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0139] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0140] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0141] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0142] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0143] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0144] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0145] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0146] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0147] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of BCRP, such labeling wouldinclude amount, frequency, and method of administration.

[0148] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0149] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0150] A therapeutically effective dose refers to that amount of activeingredient, for example BCRP or fragments thereof, antibodies of BCRP,agonists, antagonists or inhibitors of BCRP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio of toxic and therapeutic effects is thetherapeutic index, and it can be expressed as the ratio LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0151] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0152] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0153] Diagnostics

[0154] In another embodiment, antibodies which specifically bind BCRPmay be used for the diagnosis of conditions or diseases characterized byexpression of BCRP, or in assays to monitor patients being treated withBCRP, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for BCRP includemethods which utilize the antibody and a label to detect BCRP in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

[0155] A variety of protocols including ELISA, RIA, and FACS formeasuring BCRP are known in the art and provide a basis for diagnosingaltered or abnormal levels of BCRP expression. Normal or standard valuesfor BCRP expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibody to BCRP under conditions suitable for complex formation. Theamount of standard complex formation may be quantified by variousmethods, preferably by photometric means. Quantities of BCRP expressedin subject, control and disease samples from biopsied tissues arecompared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0156] In another embodiment of the invention, the polynucleotidesencoding BCRP may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, antisense RNA andDNA molecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofBCRP may be correlated with disease. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of BCRP,and to monitor regulation of BCRP levels during therapeuticintervention.

[0157] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding BCRP or closely related molecules, may be used to identifynucleic acid sequences which encode BCRP. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding BCRP, alleles, or related sequences.

[0158] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the BCRP encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring BCRP.

[0159] Means for producing specific hybridization probes for DNAsencoding BCRP include the cloning of nucleic acid sequences encodingBCRP or BCRP derivatives into vectors for the production of mRNA probes.Such vectors are known in the art, commercially available, and may beused to synthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

[0160] Polynucleotide sequences encoding BCRP may be used for thediagnosis of disorders which are associated with expression of BCRP.Examples of such conditions or diseases include, but are not limited to,cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,sarcoma, and teratocarcinoma and particularly cancers of the adrenalgland, bladder, bone, brain, breast, cervix, esophagus, gastrointestinaltract, heart, kidney, liver, lung, ovaries, pancreas, parathyroid,pituitary gland, prostate, salivary gland, spleen, stomach, thymus,thyroid, testes, and uterus; and disorders of cell growth andproliferation such as adult respiratory distress syndrome, allergies,asthma, atherosclerosis, arteriosclerosis, bronchitis, dermatomyositis,neurofibromatosis, polymyositis, rheumatoid arthritis, Alzheimer'sdisease, heart attacks, osteoarthritis, osteoporosis, Parkinson'sdisease, stroke, damage to cells such as heart muscle, and nerve cellscaused by ischemia, free radicals, and toxins, and wound healing. Thepolynucleotide sequences encoding BCRP may be used in Southern ornorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; or in dip stick, pin, ELISA or chip assays utilizingfluids or tissues from patient biopsies to detect altered BCRPexpression. Such qualitative or quantitative methods are well known inthe art.

[0161] In a particular aspect, the nucleotide sequences encoding BCRPmay be useful in assays that detect activation or induction of variouscancers, particularly those mentioned above. The nucleotide sequencesencoding BCRP may be labeled by standard methods, and added to a fluidor tissue sample from a patient under conditions suitable for theformation of hybridization complexes. After a suitable incubationperiod, the sample is washed and the signal is quantitated and comparedwith a standard value. If the amount of signal in the biopsied orextracted sample is significantly altered from that of a comparablecontrol sample, the nucleotide sequences have hybridized with nucleotidesequences in the sample, and the presence of altered levels ofnucleotide sequences encoding BCRP in the sample indicates the presenceof the associated disease. Such assays may also be used to evaluate theefficacy of a particular therapeutic treatment regimen in animalstudies, in clinical trials, or in monitoring the treatment of anindividual patient.

[0162] In order to provide a basis for the diagnosis of diseaseassociated with expression of BCRP, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes BCRP, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

[0163] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0164] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0165] Additional diagnostic uses for oligonucleotides designed from thesequences encoding BCRP may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation (5′→3′) and another with antisense(3′←5′), employed under optimized conditions for identification of aspecific gene or condition. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for detection and/or quantitation of closelyrelated DNA or RNA sequences.

[0166] Methods which may also be used to quantitate the expression ofBCRP include radiolabeling or biotinylating nucleotides, coamplificationof a control nucleic acid, and standard curves onto which theexperimental results are interpolated (Melby, P. C. et al. (1993) J.Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.212:229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor colorimetric response gives rapid quantitation.

[0167] In another embodiment of the invention, the nucleic acidsequences which encode BCRP may also be used to generate hybridizationprobes which are useful for mapping the naturally occurring genomicsequence. The sequences may be mapped to a particular chromosome or to aspecific region of the chromosome using well known techniques. Suchtechniques include FISH, FACS, or artificial chromosome constructions,such as yeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

[0168] FISH (as described in Verma et al. (1988) Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York, N.Y.) may becorrelated with other physical chromosome mapping techniques and geneticmap data. Examples of genetic map data can be found in the 1994 GenomeIssue of Science (265:1981f). Correlation between the location of thegene encoding BCRP on a physical chromosomal map and a specific disease,or predisposition to a specific disease, may help delimit the region ofDNA associated with that genetic disease. The nucleotide sequences ofthe subject invention may be used to detect differences in genesequences between normal, carrier, or affected individuals.

[0169] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (Gatti, R. A.et al. (1988) Nature 336:577-580), any sequences mapping to that areamay represent associated or regulatory genes for further investigation.The nucleotide sequence of the subject invention may also be used todetect differences in the chromosomal location due to translocation,inversion, etc. among normal, carrier, or affected individuals.

[0170] In another embodiment of the invention, BCRP, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes between BCRPand the agent being tested may be measured.

[0171] Another technique for drug screening which may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication WO84/03564. In this method, as applied to BCRP, largenumbers of different small test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The testcompounds are reacted with BCRP, or fragments thereof, and washed. BoundBCRP is then detected by methods well known in the art. Purified BCRPcan also be coated directly onto plates for use in the aforementioneddrug screening techniques. Alternatively, non-neutralizing antibodiescan be used to capture the peptide and immobilize it on a solid support.

[0172] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding BCRPspecifically compete with a test compound for binding BCRP. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with BCRP.

[0173] In additional embodiments, the nucleotide sequences which encodeBCRP may be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

[0174] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0175] I cDNA Library Construction

[0176] BRAITUT08

[0177] The BRAITUT08 cDNA library was constructed from brain tumortissue. The frozen tissue was homogenized and lysed using a PT3000homogenizer (Brinkmann Instruments, Westbury, N.J.) in guanidiniumisothiocyanate solution. The lysate was centrifuged over a 5.7 M CsClcushion using a Beckman SW28 rotor in a Beckman L8-70M ultracentrifuge(Beckman Instruments) for 18 hours at 25,000 rpm at ambient temperature.The RNA was extracted with acid phenol pH 4.7, precipitated using 0.3 Msodium acetate and 2.5 volumes of ethanol, resuspended in RNAse-freewater, and DNase treated at 37° C. The RNA extraction and precipitationwere repeated as before. The mRNA was isolated with the OLIGOTEX mRNApurification kit (QIAGEN, Inc.; Chatsworth, Calif.) and used toconstruct the cDNA library.

[0178] The mRNA was handled according to the recommended protocols inthe SUPERSCRIPT plasmid system (Gibco BRL).

[0179] BRAITUT08 cDNAs were fractionated on a SEPHAROSE CL4B column(Pharmacia), and those cDNAs exceeding 400 bp were ligated into pINCY I.The plasmid pINCY I was subsequently transformed into DH5α competentcells (Gibco BRL).

[0180] HNT2RAT01

[0181] The HNT2RAT01 cDNA library was constructed at Stratagene(STR937231), using RNA isolated from the hNT2 cell line derived from ahuman teratocarcinoma that exhibited properties characteristic of acommitted neuronal precursor at an early stage of development. Cellswere treated with 10 μM retinoic acid for 24 hours to create cells whosedifferentiation process may have commenced as described in Andrews, P.W. (1984) Dev. Biol., 103:285-293. First strand cDNA synthesis wasaccomplished using an oligo d(T) primer/linker which also contained anXhoI restriction site. Second strand synthesis was performed using acombination of DNA polymerase I, E. coli ligase and RNase H, followed bythe addition of an EcoRI adaptor to the blunt ended cDNA. The EcoRIadapted, double-stranded cDNA was then digested with XhoI restrictionenzyme and fractionated to obtain sequences which exceeded 800 bp insize. The cDNAs were inserted into the LAMBDAZAP vector system(Stratagene); and the vector which contains the PBLUESCRIPT phagemid(Stratagene) was transformed into E. coli host cells strain XL1-BLUEMRF(Stratagene).

[0182] The phagemid forms of individual cDNA clones were obtained by thein vivo excision process. Enzymes from both PBLUESCRIPT and acotransformed f1 helper phage nicked the DNA, initiated new DNAsynthesis, and created the smaller, single-stranded circular phagemidmolecules which contained the cDNA insert. The phagemid DNA wasreleased, purified, and used to reinfect fresh host cells (SOLR,Stratagene). Presence of the phagemid which carries the gene forβ-lactamase allowed transformed bacteria to grow on medium containingampicillin.

[0183] KERANOT01

[0184] The keratinocyte culture used for the KERANOT01 libraryconstruction was derived from the leg skin of 22-week male fetus (Lot#CC2503; 2859-1) obtained from Clonetics Corp, San Diego Calif.). Thecells were washed twice in phosphate buffered saline and lysedimmediately in a buffer containing guanidinium isothiocyanate. Thelysate was extracted twice with phenol chloroform and centrifuged over aCsCl cushion using a Beckman SW28 rotor and a Beckman L8-70Multracentrifuge (Beckman Instruments). The poly A+RNA was precipitatedusing 0.3 M sodium acetate and 2.5 volumes of ethanol, resuspended inwater, DNase treated for 15 min at 37° C., and isolated using theOLIGOTEX mRNA purification kit (QIAGEN, Inc.; Chatsworth, Calif.).

[0185] First strand cDNA synthesis and isolation of the phagemid formsof individual cDNA clones was accomplished using the procedure describedabove for the HNT2RAT01 cDNA library.

[0186] II Isolation and Sequencing of cDNA Clones

[0187] BRAITUT08

[0188] Plasmid DNA was released from the cells and purified using theR.E.A.L. PREP 96 plasmid purification kit (QIAGEN, Inc.). Therecommended protocol was employed except for the following changes: 1)the bacteria were cultured in 1 ml of sterile Terrific Broth (GIBCO/BRL)with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) afterinoculation, the cultures were incubated for 19 hours and at the end ofincubation, the cells were lysed with 0.3 ml of lysis buffer; and 3)following isopropanol precipitation, the plasmid DNA pellet wasresuspended in 0.1 ml of distilled water. After the last step in theprotocol, samples were transferred to a 96-well block for storage at 4°C.

[0189] HNT2RAT01, KERANOT01

[0190] Plasmid DNA was released from the cells and purified using theMINPREP DNA purification kit (Advanced Genetic Technologies Corporation,Gaithersburg Md.). This kit consists of a 96 well block with reagentsfor 960 purifications. The recommended protocol was employed except forthe following changes: 1) the 96 wells were each filled with only 1 mlof sterile Terrific Broth (GIBCO/BRL, Gaithersburg Md.) withcarbenicillin at 25 mg/L and glycerol at 0.4%; 2) the bacteria werecultured for 24 hours after the wells were inoculated and then lysedwith 60 μl of lysis buffer; 3) a centrifugation step employing theBeckman GS-6R @2900 rpm for 5 min was performed before the contents ofthe block were added to the primary filter plate; and 4) the optionalstep of adding isopropanol to TRIS buffer was not routinely performed.After the last step in the protocol, samples were transferred to aBeckman 96-well block for storage.

[0191] Alternative methods of purifying plasmid DNA include the use ofthe MAGIC MINIPREPS DNA purification system (Promega, Madison Wis.) orQIAWELL-8 Plasmid, QIAWELL PLUS DNA and QIAWELL ULTRA DNA purificationsystems (QIAGEN Chatsworth Calif.).

[0192] All of the cDNAs were sequenced by the method of Sanger, F, andA. R. Coulson (1975; J Mol Biol 94:441f), using a MICROLAB 2200 liquidtransfer system (Hamilton, Reno Nev.) in combination with four PTC200thermal cyclers (MJ Research, Watertown Mass.) and 377 or 373 DNAsequencing systems (Perkin Elmer) and the reading frame was determined.

[0193] III Homology Searching of cDNA Clones and Their Deduced Proteins

[0194] The nucleotide sequences of the Sequence Listing or amino acidsequences deduced from them were used as query sequences againstdatabases such as GenBank, SwissProt, BLOCKS, and Pima II. Thesedatabases which contain previously identified and annotated sequenceswere searched for regions of homology (similarity) using BLAST, whichstands for Basic Local Alignment Search Tool (Altschul, S. F. (1993) J.Mol. Evol. 36:290-300; Altschul et al. (1990) J. Mol. Biol.215:403-410).

[0195] BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs which may be of prokaryotic(bacterial) or eukaryotic (animal, fungal or plant) origin. Otheralgorithms such as the one described in Smith R F and T F Smith (1992;Protein Engineering 5:35-51), incorporated herein by reference, can beused when dealing with primary sequence patterns and secondary structuregap penalties. As disclosed in this application, the sequences havelengths of at least 49 nucleotides, and no more than 12% uncalled bases(where N is recorded rather than A, C, G, or T).

[0196] The BLAST approach, as detailed in Karlin, S. and S. F. Altschul(1993; Proc Nat. Acad. Sci. 90:5893-3) and incorporated herein byreference, searches for matches between a query sequence and a databasesequence, to evaluate the statistical significance of any matches found,and to report only those matches which satisfy the user-selectedthreshold of significance. In this application, threshold was set at10⁻25 for nucleotides and 10⁻¹⁴ for peptides.

[0197] Incyte nucleotide sequences were searched against the GenBankdatabases for primate (pri), rodent (rod), and mammalian sequences(mam), and deduced amino acid sequences from the same clones aresearched against GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp) and eukaryote (eukp), for homology. The relevantdatabase for a particular match were reported as a GIxxx±p (where xxx ispri, rod, etc and if present, p=peptide). Product score, the calculationof which is shown below, was used to determine the electronicstringency. For an exact match, product score was set at 70 with aconservative lower limit set at approximately 40 (1-2% error due touncalled bases).

[0198] IV Northern Analysis

[0199] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound (Sambrook et al., supra).

[0200] Analogous computer techniques using BLAST (Altschul, S. F. 1993and 1990, supra) are used to search for identical or related moleculesin nucleotide databases such as GenBank or the LIFESEQ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

[0201] The basis of the search is the product score which is defined as:$\frac{\% \quad {sequence}\quad {identity} \times {maximum}\quad {BLAST}\quad {score}}{100}$

[0202] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

[0203] The results of northern analysis are reported as a list oflibraries in which the transcript encoding BCRP occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0204] V Extension of BCRP-Encoding Polynucleotides

[0205] Nucleic acid sequence of Incyte clone 1383303 (SEQ ID NO:2),Incyte clone 484722 (SEQ ID NO:4), or Incyte clone 454790 (SEQ ID NO:6)is used to design oligonucleotide primers for extending a partialnucleotide sequence to full length or for obtaining 5′ or 3′, intron orother control sequences from genomic libraries. One primer issynthesized to initiate extension in the antisense direction (XLR) andthe other is synthesized to extend sequence in the sense direction(XLF). Primers are used to facilitate the extension of the knownsequence “outward” generating amplicons containing new, unknownnucleotide sequence for the region of interest. The initial primers aredesigned from the cDNA using OLIGO 4.06 software (National Biosciences),or another appropriate program, to be 22-30 nucleotides in length, tohave a GC content of 50% or more, and to anneal to the target sequenceat temperatures about 68°-72° C. Any stretch of nucleotides which wouldresult in hairpin structures and primer-primer dimerizations is avoided.

[0206] The original, selected cDNA libraries, or a human genomic libraryare used to extend the sequence; the latter is most useful to obtain 5′upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0207] By following the instructions for the XL-PCR kit (Perkin Elmer)and thoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the PTC200 thermal cycler (M. J. Research, Watertown,Mass.) and the following parameters: Step 1 94° C. for 1 min (initialdenaturation) Step 2 65° C. for 1 min Step 3 68° C. for 6 min Step 4 94°C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C. for 7 min Step 7Repeat step 4-6 for 15 additional cycles Step 8 94° C. for 15 sec Step 965° C. for 1 min Step 10 68° C. for 7:15 min Step 11 Repeat step 8-10for 12 cycles Step 12 72° C. for 8 min Step 13 40° C. (and holding)

[0208] A 5-10 μl aliquot of the reaction mixture is analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products are selected and removedfrom the gel. Further purification involves using a commercial gelextraction method such as QIAQUICK (QIAGEN Inc., Chatsworth, Calif.).After recovery of the DNA, Klenow enzyme is used to trimsingle-stranded, nucleotide overhangs creating blunt ends whichfacilitate religation and cloning.

[0209] After ethanol precipitation, the products are redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook et al., supra)containing 2×Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2×Carb mediumplaced in an individual well of an appropriate, commercially-available,sterile 96-well microtiter plate. The following day, 5 μl of eachovernight culture is transferred into a non-sterile 96-well plate andafter dilution 1:10 with water, 5 μl of each sample is transferred intoa PCR array.

[0210] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionare added to each well. Amplification is performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7 4° C. (andholding)

[0211] Aliquots of the PCR reactions are run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products arecompared to the original partial cDNAs, and appropriate clones areselected, ligated into plasmid, and sequenced.

[0212] VI Labeling and Use of Hybridization Probes

[0213] Hybridization probes derived from SEQ ID NO:2 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 μCi of [γ-³²P] adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DUPONT NEN, Boston, Mass.). Thelabeled oligonucleotides are substantially purified using a SEPHADEXG-25 superfine resin column (Pharmacia & Upjohn). A portion containing10⁷ counts per minute of each of the sense and antisenseoligonucleotides is used in a typical membrane based hybridizationanalysis of human genomic DNA digested with one of the followingendonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II; DUPONTNEN).

[0214] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (Nytran Plus, Schleicher &Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Kodak, Rochester, N.Y.) is exposed to the blots, or the blots areexposed to a PHOSPHOIMAGER cassette (Molecular Dynamics, Sunnyvale,Calif.), hybridization patterns are compared visually.

[0215] VIII Complementary Polynucleotides

[0216] Sequence complementary to the BCRP-encoding sequence, or any partthereof, is used to decrease or inhibit expression of naturallyoccurring BCRP. Although use of oligonucleotides comprising from about15 to about 30 base-pairs is described, essentially the same procedureis used with smaller or larger sequence fragments. Appropriateoligonucleotides are designed using OLIGO 4.06 software and the codingsequence of BCRP, SEQ ID NO:2. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5′ sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the BCRP-encoding transcript.

[0217] VIII Expression of BCRP

[0218] Expression of BCRP is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector is used to express BCRP in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

[0219] Induction of an isolated, transformed bacterial strain with IPTGusing standard methods produces a fusion protein which consists of thefirst eight residues of β-galactosidase, about 5 to 15 residues oflinker, and the full length protein. The signal residues direct thesecretion of BCRP into the bacterial growth media which can be useddirectly in the following assay for activity.

[0220] IX Demonstration of BCRP Activity

[0221] BCRP activity may be demonstrated by transfection of a mammaliancell line such as BW5147, Sp6, MEL1, or P815 (ATCC) with eukaryoticexpression vectors encoding BCRP Eukaryotic expression vectors arecommercially available, and the techniques to introduce them into cellsare well known to those skilled in the art. The cells are incubated for48-72 hours after transformation under conditions appropriate for thecell line to allow expression and accumulation of BCRP. The transformedcells are stimulated by cross-linking the surface receptors by addingeither anti-IgM, anti-IgD (Santa Cruz Biotechnology, Inc., Santa Cruz,Calif.), or both to the cell culture media. Cells are incubated in thepresence of antibody and assayed for apoptosis by the TUNEL assay(Boehringer Mannheim Corp., Indianapolis, Ind., USA), or for cellproliferation, using a BrdU cell proliferation ELISA (BoehringerMannheim). The results are evaluated by comparison with mock-transfectedand unstimulated cell controls.

[0222] X Production of BCRP Specific Antibodies

[0223] BCRP that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 is analyzed using DNASTARsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopolypeptide is synthesized and used to raiseantibodies by means known to those of skill in the art. Selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions, is described by Ausubel et al. (supra), and others.

[0224] Typically, the oligopeptides are 15 residues in length,synthesized using an Applied Biosystems 431A peptide synthesizer usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma,St. Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimideester (MBS; Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radioiodinated, goat anti-rabbitIgG.

[0225] XI Purification of Naturally Occurring BCRP Using SpecificAntibodies

[0226] Naturally occurring or recombinant BCRP is substantially purifiedby immunoaffinity chromatography using antibodies specific for BCRP. Animmunoaffinity column is constructed by covalently coupling BCRPantibody to an activated chromatographic resin, such as CnBr-activatedSEPHAROSE (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

[0227] Media containing BCRP is passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of BCRP (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/BCRP binding (eg, a buffer of pH 2-3 or a high concentration ofa chaotrope, such as urea or thiocyanate ion), and BCRP is collected.

[0228] XII Identification of Molecules Which Interact with BCRP

[0229] BCRP or biologically active fragments thereof are labeled with¹²⁵I Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled BCRP, washed and any wells withlabeled BCRP complex are assayed. Data obtained using differentconcentrations of BCRP are used to calculate values for the number,affinity, and association of BCRP with the candidate molecules.

[0230] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 10 1 240 PRT Homo sapiens misc_feature Incyte ID No 1383303 1 Met SerLeu Gln Trp Thr Ala Val Ala Thr Phe Leu Tyr Ala Glu 1 5 10 15 Val PheVal Val Leu Leu Leu Cys Ile Pro Phe Ile Ser Pro Lys 20 25 30 Arg Trp GlnLys Ile Phe Ser Phe Asn Val Trp Gly Lys Ile Ala 35 40 45 Thr Phe Trp AsnLys Ala Phe Leu Thr Ile Ile Ile Leu Leu Ile 50 55 60 Val Leu Phe Leu AspAla Val Arg Glu Val Arg Lys Tyr Ser Ser 65 70 75 Val His Thr Ile Glu LysSer Ser Thr Ser Arg Pro Asp Ala Tyr 80 85 90 Glu His Thr Gln Met Lys LeuPhe Arg Ser Gln Arg Asn Leu Tyr 95 100 105 Ile Ser Gly Phe Ser Leu PhePhe Trp Leu Val Leu Arg Arg Leu 110 115 120 Val Thr Leu Ile Thr Gln LeuAla Lys Glu Leu Ser Asn Lys Gly 125 130 135 Val Leu Lys Thr Gln Ala GluAsn Thr Asn Lys Ala Ala Lys Lys 140 145 150 Phe Met Glu Glu Asn Glu LysLeu Lys Arg Ile Leu Lys Ser His 155 160 165 Gly Lys Asp Glu Glu Cys ValLeu Glu Ala Glu Asn Lys Lys Leu 170 175 180 Val Glu Asp Gln Glu Lys LeuLys Thr Glu Leu Arg Lys Thr Ser 185 190 195 Asp Ala Leu Ser Lys Ala GlnAsn Asp Val Met Glu Met Lys Met 200 205 210 Gln Ser Glu Arg Leu Ser LysGlu Tyr Asp Gln Leu Leu Lys Glu 215 220 225 His Ser Leu Gln Asp Arg LeuGlu Arg Gly Asn Lys Lys Arg Leu 230 235 240 2 946 DNA Homo sapiensmisc_feature Incyte ID No 1383303 2 tgctgtggga gagttcggtt gctgcggcggggcctgcacg ttgactgtgg gaaactcgga 60 aacaagctca catcttcctg tgggaaaccttctagcaaca ggatgagtct gcagtggact 120 gcagttgcca ccttcctcta tgcggaggtctttgttgtgt tgcttctctg cattcccttc 180 atttctccta aaagatggca gaagattttttcatttaatg tctggggtaa aattgcaact 240 ttttggaaca aggctttcct taccattatcatcctattga ttgttctatt tctagatgct 300 gtgagagaag taaggaaata ttcctcagttcataccattg agaagagctc caccagcaga 360 cctgatgcct atgaacacac acagatgaaactttttaggt ctcaaagaaa tctttacatt 420 tctggatttt ccctattttt ttggctagttttgagacgtc tggttacgct tattactcaa 480 ctggcaaaag aactgtcaaa caaaggtgtacttaaaactc aagcagaaaa tactaacaag 540 gctgccaaaa aatttatgga agaaaacgaaaaactaaaaa ggattttgaa aagccatggt 600 aaagatgaag aatgtgtttt ggaagcagaaaataaaaaac tagtagaaga ccaggagaaa 660 ctgaaaactg aattaaggaa gacttcagatgccctttcta aggcacaaaa tgatgtgatg 720 gaaatgaaga tgcagtcaga gagactttcgaaagaatatg atcaactcct gaaagaacac 780 tctgaacttc aggatcgttt agaaagaggcaacaagaaaa gactgtgaac tttataaaag 840 acacttgcaa tatactgtgt caaaatgataattttgttat gttagcctct agaaaattta 900 agttcagaaa aatgcactat gaccggttcgtaattttttt aatgcc 946 3 299 PRT Homo sapiens misc_feature Incyte ID No484722 3 Met Ala Gln Asn Leu Lys Asp Leu Ala Gly Arg Leu Pro Ala Gly 1 510 15 Pro Arg Gly Met Gly Thr Ala Leu Lys Leu Leu Leu Gly Ala Gly 20 2530 Ala Val Ala Tyr Gly Val Arg Glu Ser Val Phe Thr Val Glu Gly 35 40 45Gly His Arg Ala Ile Phe Phe Asn Arg Ile Gly Gly Val Gln Gln 50 55 60 AspThr Ile Leu Ala Glu Gly Leu His Phe Arg Ile Pro Trp Phe 65 70 75 Gln TyrPro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Lys Ile 80 85 90 Phe Ser ProThr Gly Phe Lys Asp Leu Gln Met Val Asn Ile Xaa 95 100 105 Leu Arg ValLeu Ser Xaa Pro Asn Xaa Gln Glu Leu Xaa Ser Met 110 115 120 Tyr Gln ArgLeu Gly Val Asp Tyr Glu Glu Arg Val Leu Pro Ser 125 130 135 Ile Xaa XaaXaa Val Leu Lys Ser Val Val Ala Lys Phe Asn Ala 140 145 150 Ser Gln LeuIle Thr Gln Arg Ala Gln Val Ser Leu Leu Ile Arg 155 160 165 Arg Glu LeuThr Glu Arg Ala Lys Asp Phe Ser Leu Ile Leu Asp 170 175 180 Asp Val AlaIle Thr Glu Leu Ser Phe Ser Arg Glu Tyr Thr Ala 185 190 195 Ala Val GluAla Lys Gln Val Ala Gln Gln Glu Ala Gln Arg Ala 200 205 210 Gln Phe LeuVal Glu Lys Ala Lys Gln Glu Gln Arg Gln Lys Ile 215 220 225 Val Gln AlaGlu Gly Glu Ala Glu Ala Ala Lys Met Leu Gly Glu 230 235 240 Ala Leu SerLys Asn Pro Gly Tyr Ile Lys Leu Arg Lys Ile Arg 245 250 255 Ala Ala GlnAsn Ile Xaa Lys Thr Ile Ala Thr Ser Gln Asn Arg 260 265 270 Ile Tyr LeuThr Ala Asp Asn Leu Val Leu Asn Leu Gln Asp Glu 275 280 285 Ser Phe ThrArg Gly Ser Asp Ser Leu Ile Lys Gly Lys Lys 290 295 4 1223 DNA Homosapiens misc_feature Incyte ID No 484722 4 gggagggttt caaagggagcgcacttccgc tgccctttct ttcgccagcc ttacgggccc 60 gaaccctcgt gtgaagggtgcagtacctaa gccggagcgg ggtagaggcg ggccggcacc 120 cccttctgac ctccagtgccgccggcctca agatcagaca tggcccagaa cttgaaggac 180 ttggcgggac ggctgcccgccgggccccgg ggcatgggca cggccctgaa gctgttgctg 240 ggggccggcg ccgtggcctacggtgtgcgc gaatctgtgt tcaccgtgga aggcgggcac 300 agagccatct tcttcaatcggatcggtgga gtgcagcagg acactatcct ggccgagggc 360 cttcacttca ggatcccttggttccagtac cccattatct atgacattcg ggccagacct 420 cgaaaaatct tctcccctacagggttcaaa gacctacaga tggtgaatat ctncctgcga 480 gtgttgtctn gacccaatgntcaggagctt nctagcatgt accagcggct aggggtggac 540 tacgaggaac gagtgttgccgtccattkty aamgrggtgc tcaagagtgt ggtggccaag 600 ttcaatgcct cacagctgatcacccagcgg gcccaggtat ccctgttgat ccgccgggag 660 ctgacagaga gggccaaggacttcagcctc atcctggatg atgtggccat cacagagctg 720 agctttagcc gagagtacacagctgctgta gaagccaaac aagtggccca gcaggaggcc 780 cagcgggccc aattcttggtagaaaaagca aagcaggaac agcggcagaa aattgtgcag 840 gccgagggtg aggccgaggctgccaagatg cttggagaag cactgagcaa gaaccctggc 900 tacatcaaac ttcgcaagattcgagcagcc cagaatatct ycaagacgat cgccacatca 960 cagaatcgta tctatctcacagctgacaac cttgtgctga acctacagga tgaaagtttc 1020 accaggggaa gtgacagcctcatcaagggt aagaaatgag cctagtcacc aagaactcca 1080 cccccagagg aagtggatctrcttctrcag tttttgagga gccagccagg ggtncagcac 1140 agmcctaccc cggccyagtatcatgcgatg gtcccccaca acggtttcct gaaccctttt 1200 ggattaagga agactnaagatag 1223 5 299 PRT Homo sapiens misc_feature Incyte ID No 454790 5 MetAla Gln Asn Leu Lys Asp Leu Ala Gly Arg Leu Pro Ala Gly 1 5 10 15 ProArg Gly Met Gly Thr Ala Leu Lys Leu Leu Leu Gly Ala Gly 20 25 30 Ala ValAla Tyr Gly Val Arg Glu Ser Val Phe Thr Val Glu Gly 35 40 45 Gly His ArgAla Ile Phe Phe Asn Arg Ile Gly Gly Val Gln Gln 50 55 60 Asp Thr Ile LeuAla Glu Gly Leu His Phe Arg Ile Pro Trp Phe 65 70 75 Gln Tyr Pro Ile IleTyr Asp Ile Arg Ala Arg Pro Arg Lys Ile 80 85 90 Ser Ser Pro Thr Gly SerLys Asp Leu Gln Met Val Asn Ile Ser 95 100 105 Leu Arg Val Leu Ser ArgPro Asn Ala Gln Glu Leu Pro Ser Met 110 115 120 Tyr Gln Arg Leu Gly LeuAsp Tyr Glu Glu Arg Val Leu Pro Ser 125 130 135 Ile Val Asn Glu Val LeuLys Ser Val Val Ala Lys Phe Asn Ala 140 145 150 Ser Gln Leu Ile Xaa GlnArg Ala Gln Val Ser Leu Leu Ile Arg 155 160 165 Arg Glu Leu Thr Glu ArgAla Lys Asp Phe Ser Leu Ile Leu Asp 170 175 180 Asp Val Ala Ile Thr GluLeu Ser Phe Ser Arg Glu Tyr Thr Ala 185 190 195 Ala Val Glu Ala Lys GlnVal Ala Gln Gln Glu Ala Gln Arg Ala 200 205 210 Gln Phe Leu Val Glu LysAla Lys Gln Glu Gln Arg Gln Lys Ile 215 220 225 Val Gln Ala Glu Gly GluAla Glu Ala Ala Lys Met Leu Gly Glu 230 235 240 Ala Leu Ser Lys Asn ProGly Tyr Ile Lys Leu Arg Lys Ile Arg 245 250 255 Ala Ala Gln Asn Ile SerLys Thr Ile Ala Thr Ser Gln Asn Arg 260 265 270 Ile Tyr Leu Thr Ala AspAsn Leu Val Leu Asn Leu Gln Asp Glu 275 280 285 Ser Phe Thr Arg Gly SerAsp Ser Leu Ile Lys Gly Lys Lys 290 295 6 1212 DNA Homo sapiensmisc_feature Incyte ID No 454790 6 gcacttccgc tgccctttct ttcgccagccttacgggccc gaaccctcgt gtgaagggtg 60 cagtacctaa gccggagcgg ggtagaggcgggccggcacc cccttctgac ctccagtgcc 120 gccggcctca agatcagaca tggcccagaacttgaaggac ttggcgggac ggctgcccgc 180 cgggccccgg ggcatgggca cggccctgaagctgttgctg ggggccggcg ccgtggccta 240 cggtgtgcgc gaatctgtgt tcaccgtggaaggcgggcac agagccatct tcttcaatcg 300 gatcggtgga gtgcagcagg acactatcctggccgagggc cttcacttca ggatcccttg 360 gttccagtac cccattatct atgacattcgggccagacct cgaaaaatct cctcccctac 420 aggctccaaa gacctacaga tggtgaatatctccctgcga gtkttgtctc gacccaatgc 480 tcaggagctt cctagcatgt accagcgcctagggctggac tacgaggaac gagtgttgcc 540 gtccattgtc aacgaggtgc tcaagagtgtggtggccaag ttcaatgcct cacagctgat 600 camccagcgg gcccaggtat ccctgttgatccgccgggag ctgacagaga gggccaagga 660 cttcagcctc atcctggatg atgtggccatcacagagctg agytttagcc gagagtacac 720 agctgctgta gaagccaaac aagtggcccagcaggaggcc cagcgggccc aattcttggt 780 agaaaaagca aagcaggaac agcggcagaaaattgtgcag gccgagggtg aggccgaggc 840 tgccaagatg cttggagaag cactgagcaagaaccctggc tacatcaaac ttcgcaagat 900 tcgagcagcc cagaatatct ccaagacgatcgccacatca cagaatcgta tctatctcac 960 agctgacaac cttgtgctga acctacaggatgaaagtttc accaggggaa gtgacagcct 1020 catcaagggt aagaaatgag cctagtcaccaagaactcca cccccagagg aagtggatct 1080 gcttcttcca gtttttgagg agccagccaggggtccagca cagccctacc ccgccccagt 1140 atcatgcgat ggtcccccaa aacggtttcctgaacccctc ttggattaag gaagactgaa 1200 gactagcccc nc 1212 7 239 PRT Musmusculus misc_feature GenBank ID No g541730 7 Met Thr Ile Gln Trp AlaAla Val Ala Ser Phe Leu Tyr Ala Glu 1 5 10 15 Ile Gly Leu Ile Leu LeuPhe Cys Leu Pro Phe Ile Pro Pro Gln 20 25 30 Arg Trp Gln Lys Ile Phe SerPhe Ser Val Trp Gly Lys Ile Ala 35 40 45 Ser Phe Trp Asn Lys Ala Phe LeuThr Ile Ile Ile Leu Leu Ile 50 55 60 Ile Leu Phe Leu Asp Ala Val Arg GluVal Arg Lys Tyr Ser Ser 65 70 75 Thr Asn Val Val Glu Lys Asn Ser Ala IleArg Pro Ser Ala Phe 80 85 90 Glu His Thr Gln Met Lys Leu Phe Arg Ser GlnArg Asn Leu Tyr 95 100 105 Ile Ser Gly Phe Ser Leu Phe Phe Trp Leu ValLeu Arg Arg Leu 110 115 120 Val Thr Leu Ile Thr Gln Leu Ala Lys Glu IleAla Asn Lys Gly 125 130 135 Val Leu Lys Ile Gln Ala Glu Asn Thr Asn LysAla Ala Lys Lys 140 145 150 Phe Met Glu Asn Glu Lys Leu Lys Leu Gly LeuArg Asn Asp Asn 155 160 165 Ala Glu Glu His Leu Leu Glu Ala Glu Asn LysLys Leu Ile Glu 170 175 180 Ser Lys Glu Asn Leu Lys Thr Glu Leu Lys LysAla Ser Asp Ala 185 190 195 Leu Leu Lys Ala Gln Asn Asp Val Met Thr MetLys Ile Gln Ser 200 205 210 Glu Arg Leu Ser Lys Glu Tyr Asp Arg Leu LeuLys Glu His Ser 215 220 225 Glu Leu Gln Asn Arg Leu Glu Lys Glu Lys LysLys Gly Leu 230 235 8 298 PRT Mus musculus misc_feature GenBank ID Nog541734 8 Met Ala Gln Asn Leu Lys Asp Leu Ala Gly Arg Leu Pro Ala Gly 15 10 15 Pro Arg Gly Met Gly Thr Ala Leu Lys Leu Leu Leu Gly Ala Gly 2025 30 Ala Val Ala Tyr Gly Val Arg Glu Ser Val Phe Thr Val Glu Gly 35 4045 Gly His Arg Ala Ile Phe Phe Asn Arg Ile Gly Gly Val Gln Gln 50 55 60Asp Thr Ile Leu Ala Glu Phe His Phe Arg Ile Pro Trp Phe Gln 65 70 75 TyrPro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Lys Ile Ser 80 85 90 Ser ProThr Gly Ser Lys Asp Leu Gln Met Val Asn Ile Ser Leu 95 100 105 Arg ValLeu Ser Arg Pro Asn Ala Gln Glu Leu Pro Ser Met Tyr 110 115 120 Gln ArgLeu Gly Leu Asp Tyr Glu Glu Arg Val Leu Pro Ser Ile 125 130 135 Val AsnGlu Val Leu Lys Ser Val Val Ala Lys Phe Asn Ala Ser 140 145 150 Gln LeuIle Thr Gln Arg Ala Gln Val Ser Leu Leu Ile Arg Arg 155 160 165 Glu LeuThr Glu Arg Ala Lys Asp Phe Ser Leu Ile Leu Asp Asp 170 175 180 Val AlaIle Thr Glu Leu Ser Phe Ser Arg Glu Tyr Thr Ala Ala 185 190 195 Val GluAla Lys Gln Val Ala Gln Gln Glu Ala Gln Arg Ala Gln 200 205 210 Phe LeuVal Glu Lys Ala Lys Gln Glu Gln Arg Gln Lys Ile Val 215 220 225 Gln AlaGlu Gly Glu Ala Glu Ala Ala Lys Met Leu Gly Glu Ala 230 235 240 Leu SerLys Asn Pro Gly Tyr Ile Lys Leu Arg Lys Ile Arg Ala 245 250 255 Ala GlnAsn Ile Ser Lys Thr Ile Ala Thr Ser Gln Asn Arg Ile 260 265 270 Tyr LeuThr Ala Asp Asn Leu Val Leu Asn Leu Gln Asp Glu Ser 275 280 285 Phe ThrArg Gly Ser Asp Ser Leu Ile Lys Gly Lys Lys 290 295 9 4 PRT Homo sapiensmisc_feature Incyte ID No 000 9 Ile Thr Ala Met 1 10 4 PRT Homo sapiensmisc_feature Incyte ID No 000 10 Asn Pro Xaa Tyr 1

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQID NO:5, b) a polypeptide comprising a naturally occurring amino acidsequence at least 90% identical to an amino acid sequence selected fromthe group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5, c) abiologically active fragment of a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3,and SEQ ID NO:5, and d) an immunogenic fragment of a polypeptide havingan amino acid sequence selected from the group consisting of SEQ IDNO:1, SEQ ID NO:3, and SEQ ID NO:5.
 2. An isolated polypeptide of claim1 comprising an amino acid sequence selected from the group consistingof SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5.
 3. An isolatedpolynucleotide encoding a polypeptide of claim
 1. 4. An isolatedpolynucleotide encoding a polypeptide of claim
 2. 5. An isolatedpolynucleotide of claim 4 comprising a polynucleotide sequence selectedfrom the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6.6. A recombinant polynucleotide comprising a promoter sequence operablylinked to a polynucleotide of claim
 3. 7. A cell transformed with arecombinant polynucleotide of claim
 6. 8. A transgenic organismcomprising a recombinant polynucleotide of claim
 6. 9. A method ofproducing a polypeptide of claim 1, the method comprising: a) culturinga cell under conditions suitable for expression of the polypeptide,wherein said cell is transformed with a recombinant polynucleotide, andsaid recombinant polynucleotide comprises a promoter sequence operablylinked to a polynucleotide encoding the polypeptide of claim 1, and b)recovering the polypeptide so expressed.
 10. A method of claim 9,wherein the polypeptide comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5. 11.An isolated antibody which specifically binds to a polypeptide ofclaim
 1. 12. An isolated polynucleotide selected from the groupconsisting of: a) a polynucleotide comprising a polynucleotide sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQID NO:6, b) a polynucleotide comprising a naturally occurringpolynucleotide sequence at least 90% identical to a polynucleotidesequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4,and SEQ ID NO:6, c) a polynucleotide complementary to a polynucleotideof a), d) a polynucleotide complementary to a polynucleotide of b), ande) an RNA equivalent of a)-d)
 13. An isolated polynucleotide comprisingat least 60 contiguous nucleotides of a polynucleotide of claim
 12. 14.A method of detecting a target polynucleotide in a sample, said targetpolynucleotide having a sequence of a polynucleotide of claim 12, themethod comprising: a) hybridizing the sample with a probe comprising atleast 20 contiguous nucleotides comprising a sequence complementary tosaid target polynucleotide in the sample, and which probe specificallyhybridizes to said target polynucleotide, under conditions whereby ahybridization complex is formed between said probe and said targetpolynucleotide or fragments thereof, and b) detecting the presence orabsence of said hybridization complex, and, optionally, if present, theamount thereof.
 15. A method of claim 14, wherein the probe comprises atleast 60 contiguous nucleotides.
 16. A method of detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 12, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 17. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 18. Acomposition of claim 17, wherein the polypeptide comprises an amino acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3,and SEQ ID NO:5.
 19. A method for treating a disease or conditionassociated with decreased expression of functional BCRP, comprisingadministering to a patient in need of such treatment the composition ofclaim
 17. 20. A method of screening a compound for effectiveness as anagonist of a polypeptide of claim 1, the method comprising: a) exposinga sample comprising a polypeptide of claim 1 to a compound, and b)detecting agonist activity in the sample.
 21. A composition comprisingan agonist compound identified by a method of claim 20 and apharmaceutically acceptable excipient.
 22. A method for treating adisease or condition associated with decreased expression of functionalBCRP, comprising administering to a patient in need of such treatment acomposition of claim
 21. 23. A method of screening a compound foreffectiveness as an antagonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting antagonist activity in the sample.
 24. Acomposition comprising an antagonist compound identified by a method ofclaim 23 and a pharmaceutically acceptable excipient.
 25. A method fortreating a disease or condition associated with overexpression offunctional BCRP, comprising administering to a patient in need of suchtreatment a composition of claim
 24. 26. A method of screening for acompound that specifically binds to the polypeptide of claim 1, themethod comprising: a) combining the polypeptide of claim 1 with at leastone test compound under suitable conditions, and b) detecting binding ofthe polypeptide of claim 1 to the test compound, thereby identifying acompound that specifically binds to the polypeptide of claim
 1. 27. Amethod of screening for a compound that modulates the activity of thepolypeptide of claim 1, the method comprising: a) combining thepolypeptide of claim 1 with at least one test compound under conditionspermissive for the activity of the polypeptide of claim 1, b) assessingthe activity of the polypeptide of claim 1 in the presence of the testcompound, and c) comparing the activity of the polypeptide of claim 1 inthe presence of the test compound with the activity of the polypeptideof claim 1 in the absence of the test compound, wherein a change in theactivity of the polypeptide of claim 1 in the presence of the testcompound is indicative of a compound that modulates the activity of thepolypeptide of claim
 1. 28. A method of screening a compound foreffectiveness in altering expression of a target polynucleotide, whereinsaid target polynucleotide comprises a sequence of claim 5, the methodcomprising: a) exposing a sample comprising the target polynucleotide toa compound, under conditions suitable for the expression of the targetpolynucleotide, b) detecting altered expression of the targetpolynucleotide, and c) comparing the expression of the targetpolynucleotide in the presence of varying amounts of the compound and inthe absence of the compound.
 29. A method of assessing toxicity of atest compound, the method comprising: a) treating a biological samplecontaining nucleic acids with the test compound, b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide of claim 12 underconditions whereby a specific hybridization complex is formed betweensaid probe and a target polynucleotide in the biological sample, saidtarget polynucleotide comprising a polynucleotide sequence of apolynucleotide of claim 12 or fragment thereof, c) quantifying theamount of hybridization complex, and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 30. Amethod for a diagnostic test for a condition or disease associated withthe expression of BCRP in a biological sample, the method comprising: a)combining the biological sample with an antibody of claim 11, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex, and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 31. The antibody of claim 11, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 32. Acomposition comprising an antibody of claim 11 and an acceptableexcipient.
 33. A method of diagnosing a condition or disease associatedwith the expression of BCRP in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 32. 34. Acomposition of claim 32, wherein the antibody is labeled.
 35. A methodof diagnosing a condition or disease associated with the expression ofBCRP in a subject, comprising administering to said subject an effectiveamount of the composition of claim
 34. 36. A method of preparing apolyclonal antibody with the specificity of the antibody of claim 11,the method comprising: a) immunizing an animal with a polypeptideconsisting of an amino acid sequence selected from the group consistingof SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5, or an immunogenic fragmentthereof, under conditions to elicit an antibody response, b) isolatingantibodies from the animal, and c) screening the isolated antibodieswith the polypeptide, thereby identifying a polyclonal antibody whichspecifically binds to a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQID NO:5.
 37. A polyclonal antibody produced by a method of claim
 36. 38.A composition comprising the polyclonal antibody of claim 37 and asuitable carrier.
 39. A method of making a monoclonal antibody with thespecificity of the antibody of claim 11, the method comprising: a)immunizing an animal with a polypeptide consisting of an amino acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3,and SEQ ID NO:5, or an immunogenic fragment thereof, under conditions toelicit an antibody response, b) isolating antibody producing cells fromthe animal, c) fusing the antibody producing cells with immortalizedcells to form monoclonal antibody-producing hybridoma cells, d)culturing the hybridoma cells, and e) isolating from the culturemonoclonal antibody which specifically binds to a polypeptide comprisingan amino acid sequence selected from the group consisting of SEQ IDNO:1, SEQ ID NO:3, and SEQ ID NO:5.
 40. A monoclonal antibody producedby a method of claim
 39. 41. A composition comprising the monoclonalantibody of claim 40 and a suitable carrier.
 42. The antibody of claim11, wherein the antibody is produced by screening a Fab expressionlibrary.
 43. The antibody of claim 11, wherein the antibody is producedby screening a recombinant immunoglobulin library.
 44. A method ofdetecting a polypeptide comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5 in asample, the method comprising: a) incubating the antibody of claim 11with the sample under conditions to allow specific binding of theantibody and the polypeptide, and b) detecting specific binding, whereinspecific binding indicates the presence of a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:3, and SEQ ID NO:5 in the sample.
 45. A method of purifying apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5 from a sample,the method comprising: a) incubating the antibody of claim 11 with thesample under conditions to allow specific binding of the antibody andthe polypeptide, and b) separating the antibody from the sample andobtaining the purified polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQID NO:5.
 46. A microarray wherein at least one element of the microarrayis a polynucleotide of claim
 13. 47. A method of generating anexpression profile of a sample which contains polynucleotides, themethod comprising: a) labeling the polynucleotides of the sample, b)contacting the elements of the microarray of claim 46 with the labeledpolynucleotides of the sample under conditions suitable for theformation of a hybridization complex, and c) quantifying the expressionof the polynucleotides in the sample.
 48. An array comprising differentnucleotide molecules affixed in distinct physical locations on a solidsubstrate, wherein at least one of said nucleotide molecules comprises afirst oligonucleotide or polynucleotide sequence specificallyhybridizable with at least 30 contiguous nucleotides of a targetpolynucleotide, and wherein said target polynucleotide is apolynucleotide of claim
 12. 49. An array of claim 48, wherein said firstoligonucleotide or polynucleotide sequence is completely complementaryto at least 30 contiguous nucleotides of said target polynucleotide. 50.An array of claim 48, wherein said first oligonucleotide orpolynucleotide sequence is completely complementary to at least 60contiguous nucleotides of said target polynucleotide.
 51. An array ofclaim 48, wherein said first oligonucleotide or polynucleotide sequenceis completely complementary to said target polynucleotide.
 52. An arrayof claim 48, which is a microarray.
 53. An array of claim 48, furthercomprising said target polynucleotide hybridized to a nucleotidemolecule comprising said first oligonucleotide or polynucleotidesequence.
 54. An array of claim 48, wherein a linker joins at least oneof said nucleotide molecules to said solid substrate.
 55. An array ofclaim 48, wherein each distinct physical location on the substratecontains multiple nucleotide molecules, and the multiple nucleotidemolecules at any single distinct physical location have the samesequence, and each distinct physical location on the substrate containsnucleotide molecules having a sequence which differs from the sequenceof nucleotide molecules at another distinct physical location on thesubstrate.
 56. A polypeptide of claim 1, comprising the amino acidsequence of SEQ ID NO:1.
 57. A polypeptide of claim 1, comprising theamino acid sequence of SEQ ID NO:3.
 58. A polypeptide of claim 1,comprising the amino acid sequence of SEQ ID NO:5.
 59. A polynucleotideof claim 12, comprising the polynucleotide sequence of SEQ ID NO:2. 60.A polynucleotide of claim 12, comprising the polynucleotide sequence ofSEQ ID NO:4.
 61. A polynucleotide of claim 12, comprising thepolynucleotide sequence of SEQ ID NO:6.